CD19+ and CD14+ cells were deleted from splenocytes from Vα19iTg Cd1d1−/− mice using anti-mouse CD19 and anti-CD14 MicroBeads (Miltenyi Biotec). MAIT cells (F4/80− CD3+ MR1−tetramer+) were sorted from CD14−CD19− splenocytes by MoFlo Astrios EQ (Beckman Coulter). CD19+ splenocytes were sorted from the spleens of FcγRIIb−/−Yaa mice using anti-mouse CD19 MicroBeads. Then, 100,000 or 20,000 MAIT cells and 20,000 CD19+ cells were co-cultured in 96-well V-bottom plates (Corning) with 100 ng/mL of lipopolysaccharide (LPS; InvivoGen) for 7 days. In some experiments, 10 μg/mL of anti-mouse CD154 antibody, anti-mouse CD275 antibody, anti-mouse MR1 antibody (all from BioLegend) or 10 μM i6-FP was added.
Lipopolysaccharide lps
Manufactured by InvivoGen
Sourced in United States, France, United Kingdom
Lipopolysaccharide (LPS) is a key component of the outer membrane of Gram-negative bacteria. It functions as an endotoxin and is recognized by the innate immune system, triggering a strong inflammatory response.
Automatically generated - may contain errors
Lab products found in correlation
Pam3csk4, by InvivoGen (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Cpg a, by InvivoGen (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Resiquimod r848, by InvivoGen (4 mentions)
Flagellin, by InvivoGen (4 mentions)
Imiquimod, by InvivoGen (4 mentions)
Zymosan, by InvivoGen (3 mentions)
Poly 1 c, by InvivoGen (3 mentions)
Polyinosinic polycytidylic acid, by InvivoGen (3 mentions)
Cgamp, by InvivoGen (3 mentions)
Murine gm csf, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (3 mentions)
Tnf α, by R&D Systems (2 mentions)
Pam3cyssk4, by InvivoGen (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Nigericin, by InvivoGen (2 mentions)
66 protocols using lipopolysaccharide lps
1
Isolation and Co-culture of MAIT and B Cells
2
Mitochondrial Dysfunction in Immune Signaling
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A list of all reagents, chemicals or kits used in this study can be found in Table S1 . Lipopolysaccharide (LPS) and high molecular weight Poly(I:C) (PIC) were purchased from InvivoGen. MitoTEMPO (MT), N-acetylcysteine (NAC), antimycin A (AA), rotenone (ROT), potassium cyanide, 2-deoxyglucose (2-DG), oligomycin (OM) and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were acquired from Sigma-Aldrich while S3QEL-2 was purchased from Cedarlane. IL-1β, IL-6, IL-10, TNF-α, and CXCL10 ELISA kits were purchased from R&D Systems. The IFN-α/IFN-β 2-Plex Mouse ProcartaPlexTM Luminex Panel kit used was from Invitrogen. Tetramethylrhodamine, methyl ester (TMRM), MitoSox Red and CellROX Orange probes were from ThermoFisher. Antibodies against Complex II (SDHB) was from Abcam while antibodies recognizing SOD2 was purchased from Cell Signalling Technology. Antibodies targeting IRF3, pIRF3 (Ser385), IRF7, pIRF7 (Ser477), GPX4, Iκbα, Complexes I (NDUFB8), III (UQCRC2) and IV (COX4) were purchased from ThermoFisher.
3
Stimulation of PBMC with Diverse Antigens
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0.1 × 106 PBMCs in 100 µL of culture medium (2% penicillin/streptomycin, 1% l -glutamine, and 10% FCS in RPMI) were stimulated in 96-well round bottom plates for 16 h with the following antigens: purified protein derivative (PPD) (10 µg/mL, Serum Staten Institute, Denmark), heat-killed Listeria monocytogenes (HKLM) (TLR2 agonist; 109/mL, Invivogen), lipopolysaccharide (LPS) (TLR4 ligand; 1 µg/mL, Invivogen), CLO-75 (TLR7/8 agonist; 10 µg/mL, Invivogen); and heat-killed Streptococcus pneumonia (SP) (105 cells/mL), CA (105 cells/mL), or Escherichia coli (EC) (106 cells/mL). PMA/ionomycin (0.1 μg/1 μg/mL, Sigma, UK) was used as a positive control, and RPMI as a negative control. Supernatants were collected after the addition of 100 µL of RPMI and centrifugation at 1,500 rpm for 10 min. Supernatants were stored at −20°C until needed for cytokine analysis.
4
Lipid Characterization in Macrophage Activation
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1,2-dipalmitoyl-sn-glycero-3-phospocholine (DPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids Inc. (Birmingham, AL, USA). Lissamine Rhodamine B 1,2-dihexadecanoyl-sn-glycero-3 phosphoethanolamine (rhodamine-DHPE) was provided by ThermoFisher Scientific Co., Ltd. (USA). Disodium fluorescein was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipopolysaccharide (LPS) was provided by InvivoGen (San Diego, CA, USA), and Interferon gamma (INF-γ) was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The cell line J774A.1 was obtained from American Type Culture Collection (ATCC, USA). Fetal bovine serum (FBS) and exosome-depleted FBS were provided by Thermo Scientific (Paisley, UK). All the other reagents used in the experiments were of analytical grade (> 98%).
5
In vitro Immune Cell Stimulation
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For the in vitro infection assays, 1x105 or 1x106 HaCaT cells were seeded into a 24-well plate or a 12-well plate, respectively, and cultured to confluence at 37°C with 5% CO2 for 24 hours. The following stimulating agents were used: lipopolysaccharide (LPS) 0.5 µg/ml (Invivogen), Pam3Cys (P3C) 0.5 µg/ml (EMC microcollections GmbH), resiquimod (R848) 5 µg/ml (Enzo Life Sciences GmbH), CpG oligodeoxynucleotides (CpG) 1 µM (MWG-Biotech AG), polycytidylic acid (Poly(I:C)) 1 μg/ml (Invivogen), peptidoglycan (PGN) 10 μg/ml (Invivogen) and muramyldipeptide (MDP) 5 μg/ml.
6
Isoliquiritigenin: Modulating Inflammatory Pathways
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ILG (Isoliquiritigenin, CAS: 961‐29‐5) was purchased from Aladdin (Aladdin Bio‐Chem Technology Company, Shanghai, China). Caerulein (Cae) and dimethyl sulfoxide (DMSO) was obtained from Sigma‐Aldrich. Recombinant human TGF‐β1 and recombinant mouse IL‐4 were obtained from R&D Systems (Minneapolis, MN, USA). Lipopolysaccharide (LPS) was purchased from InvivoGen (InvivoGen, France). Sodium carboxymethyl cellulose (Na‐CMC) was obtained from Sangon Biotechnology (Shanghai, China). Early Apoptosis Detection Kit (#6592) was obtained from Cell Signaling Technology (Danvers, Massachusetts, USA).
7
Comparative study of local anesthetic effects
8
Stimulation and Cytokine Measurement
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Blood cell stimulation employed flat-bottom 6-well plates (TPP, Switzerland). Per well, 5 × 106 cells were cultured in 3 mL Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies).
The synthetic TLR ligands Pam3Cys-SK4 (10 μg/mL), polyinosine–polycytodylic acid (polyI:C) (10 μg/mL), lipopolysaccharide (LPS) (10 μg/mL) and resiquimod (R848) (10 μg/mL), all from InvivoGen, were used for stimulation of cells over 18 h. Likewise the mitogens phorbol 12-myristate 13-acetate (PMA) (200 ng/mL), ionomycin (1 μg/mL) and concanavalin A (10 μg/mL), all from Sigma-Aldrich, were used for stimulation of cells over 18 h.
Stimulation with M. bovis (live or heat-inactivated) was done at a multiplicity of infection (MOI) of 0.1, based on preliminary experiments, for 18 h.
After 14 h, 50–100 μL of cell culture supernatant was collected and frozen for further cytokine secretion measurement (multiplex immunoassay). As soon after, Brefeldin A (10 μg/mL) (ThermoFisher) was added to the medium to block cytokine secretion; incubation was extended for another 4 h, to allow the de novo cytokine synthesis measurement (flow cytometry intracellular staining).
The synthetic TLR ligands Pam3Cys-SK4 (10 μg/mL), polyinosine–polycytodylic acid (polyI:C) (10 μg/mL), lipopolysaccharide (LPS) (10 μg/mL) and resiquimod (R848) (10 μg/mL), all from InvivoGen, were used for stimulation of cells over 18 h. Likewise the mitogens phorbol 12-myristate 13-acetate (PMA) (200 ng/mL), ionomycin (1 μg/mL) and concanavalin A (10 μg/mL), all from Sigma-Aldrich, were used for stimulation of cells over 18 h.
Stimulation with M. bovis (live or heat-inactivated) was done at a multiplicity of infection (MOI) of 0.1, based on preliminary experiments, for 18 h.
After 14 h, 50–100 μL of cell culture supernatant was collected and frozen for further cytokine secretion measurement (multiplex immunoassay). As soon after, Brefeldin A (10 μg/mL) (ThermoFisher) was added to the medium to block cytokine secretion; incubation was extended for another 4 h, to allow the de novo cytokine synthesis measurement (flow cytometry intracellular staining).
9
Isolation and Differentiation of Immune Cells
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Blood samples were collected in CPDA-1 bags (Terumo, Japan). PBMCs were isolated from blood samples using Ficoll-Hypaque density gradient technique (Sigma-Aldrich, Germany).
Monocytes were isolated by adherence for 2hrs. in RPMI media with no serum or using Pan Monocyte Isolation kit (Miltenyi Biotec, Germany) according to the manufacturer instructions. Macrophages were obtained from monocytes cultured for 6 days in RPMI media (Gibco, UK) supplemented with 50 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D, UK). MDDCs were differentiated from monocytes for 6 days in RPMI media supplemented with 50 ng/ml GM-CSF and 50 ng/ml of human recombinant interleukin-4 (IL-4; R&D, UK). Cells (106 cells/ml) were stimulated for 24 hrs with 20ng/ml lipopolysaccharide (LPS; InvivoGen, USA).
Monocytes were isolated by adherence for 2hrs. in RPMI media with no serum or using Pan Monocyte Isolation kit (Miltenyi Biotec, Germany) according to the manufacturer instructions. Macrophages were obtained from monocytes cultured for 6 days in RPMI media (Gibco, UK) supplemented with 50 ng/ml human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D, UK). MDDCs were differentiated from monocytes for 6 days in RPMI media supplemented with 50 ng/ml GM-CSF and 50 ng/ml of human recombinant interleukin-4 (IL-4; R&D, UK). Cells (106 cells/ml) were stimulated for 24 hrs with 20ng/ml lipopolysaccharide (LPS; InvivoGen, USA).
10
Measuring IL-6 Responses to TLR Agonists
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DC and myoblasts were harvested and transferred (respectively 50x103 and 10x103 cells/well) to 96-well plates and incubated with LipoPolySaccharide LPS (1 μg/mL), flagellin (10 ng/mL), zymosan (1 μg/mL), CpG-A (10 μg/mL), imiquimod (0.5 μg/mL), poly (I:C) (1 μg/mL), or Pam3CSK4 (100 ng/mL), all from Invivogen, for 16 hr. IL-6 concentration was measured in culture supernatants using a Human IL-6 ELISA MAX Deluxe, according to the manufacturer’s instructions and performing 3 to 4 technical replicates per condition. The results are expressed in picograms per milliliter (pg/mL). Unless otherwise specified in figures or legends, cytokine concentrations in vitro are presented after background subtraction, obtained with cells incubated with vehicle (DMSO or water). In some experiments the TLR9 antagonist ODN TTAGGG (A151) was added 1 hr before addition of stimuli at the concentration of 1 μmol/L.
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